Ghrelin (AG) is a 28 amino acid peptide, purified and identified from rat stomach, and characterized by the presence of an n-octanoyl modification on the Ser3 residue (Ref. 1). AG is the endogenous ligand of the growth hormone secretagogue receptor (GHSR) (Refs. 2, 3) and in addition to growth-hormone-releasing properties, AG is also detected in the cardiovascular system including in the heart, vasculature and endothelial cells of large vessels, indicating that it may also influence vascular biology, vascular physiology, and atherogenesis (Refs. 4, 5, 6).
Des-acyl ghrelin (or unacylated ghrelin, UAG), the unacylated form of ghrelin, whose concentration in plasma and tissue is higher, compared to AG, fails to bind GHSR-1a and is devoid of any central activity (Ref. 7). However, UAG shares with AG many biological activities and common binding sites on several peripheral tissues. AG and UAG exhibit similar GHS-R independent biological activities, including a cytoprotective effect (Ref. 9) and an effect on adipogenesis in vivo (Ref. 10). In most, but not all, of the cells where UAG activity was evaluated, GHSR-1a is not expressed, suggesting that such pleiotropic activities shared with AG may be mediated by a yet unidentified receptor distinct from GHSR-1a.
It has been demonstrated that UAG is a biologically active peptide, particularly at the metabolic level, having notably been shown to exert anti-diabetogenic effects as described in U.S. Pat. No. 7,485,620, in U.S. patent application publication number U.S. 20080159991, in U.S. patent application publication number U.S. 20080312133 and in WO/2008/145749.
It was previously generally reported that AG and UAG act directly on cardiomyocytes to inhibit experimentally-induced cell death through activation of a survival signaling pathway (Ref. 8). AG was also shown to inhibit basal and TNF-α-induced chemotactic cytokine production and mononuclear cell adhesion in human umbilical vein endothelial cells (HUVECs) (Ref. 5). It was further reported that treatment of human microvascular endothelial cells (HMVECs) with exogenous AG significantly increased cell proliferation, migration, in vitro angiogenesis and ERK2 phosphorylation in these cells (Ref. 4). Recently, Kleinz et al. (Ref. 35) demonstrated that AG and UAG play a role in the paracrine regulation of vascular tone in humans; more specifically they showed that AG and UAG have vasodilator actions in human arteries.
Accelerated vascular disease is the major cause of death and disability in patients with diabetes. Endothelial injury is thought to represent a crucial step in initiation and progression of atherosclerotic vascular disease in diabetes setting (Ref. 11).
It was previously reported that advanced glycated end products (AGEs) contribute to impaired vascular remodeling in the diabetic setting (Ref. 12). The formation of AGEs and the production of reactive oxygen species (ROS), as a cellular response to AGE in diabetes, seem to mainly contribute to these events.
Vascular remodeling does not rely exclusively on proliferation of resident endothelial cells but also involves circulating endothelial progenitor cells (EPC). Recent data demonstrated that in patients with cardiovascular risk factors such as, but not limited to, patients with diabetes, the number of EPC is reduced and their function impaired (Refs. 13, 14, 15).
Two types of EPC have so far been described, the early and late EPC. Although they share common features, they have some distinct features with respect to morphology, proliferative potential, and in vitro functional characteristics. Unlike late EPC, early EPC do not adopt a typical endothelial phenotype in vitro but enhance neovascularization in an indirect paracrine fashion in vivo. This led to redefining these cells as circulating angiogenic cells (CAC). CAC, that are monocyte-like cells, may home from the bone-marrow into sites of neovascularization, participate in re-endothelization after vascular injury and differentiate into mature endothelial cells in situ (Ref. 16).
Compelling evidence indicates that as the cardiovascular risk factor profile increases, CAC number decreases and CAC functional activity is impaired, thus limiting CAC delivery to, for example, sites of ischemia where angiogenesis could be required. Treatment with certain cytokines induces bone-marrow (BM) mobilization of CAC which, in turn, likely protects against cardiovascular risk (Refs. 17, 18).
Oxidative stress plays a major role in vascular tissue damage and endothelial injury associated with diabetes. Mainly, the production of ROS in this setting is induced by advanced glycated end products (AGEs), notably produced from CAC.
There is an important need to design a way to improve vascular remodeling and noevascularization in patients at risk of suffering from a cardiovascular disease or suffering from a cardiovascular disease in order to prevent or to treat cardiovascular diseases. One solution is to increase CAC cell number and/or improve CAC functionality, which can be achieved by, improving their mobilization from the bone marrow, decreasing ROS production induced by AGEs, decreasing CAC senescence or apoptosis rate, and by enhancing CAC capacity to differentiate into an arterial or a venous phenotype (i.e., to form vessels in vivo).
The earlier observations that AG may have an effect on vascular dysfunction and cardio-protection led to evaluate the in vitro and in vivo effect of UAG on same, as well as to evaluate the effect of UAG on CAC biology, which notably, has not been demonstrated before.